Ligations

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Reagents

  • T4 DNA ligase
  • 10x T4 DNA Ligase Buffer
  • Deionized, sterile H2O
  • Purified, linearized vector (likely in H2O or EB)
  • Purified, linearized insert (likely in H2O or EB)

Equipment

  • Vortex

Procedure
10μL Ligation Mix
Note: Larger ligation mixes are also commonly used

  • 1.0 μL 10X T4 ligase buffer
  • 6:1 molar ratio of insert to vector (~10ng vector)
  • Add (8.5 - vector and insert volume)μl ddH2O
  • 0.5 μL T4 Ligase


Calculating Insert Amount

Insert.jpg